PO-01-178 MAPPING OF PATHOGENIC VARIANT HOTSPOTS ON THE STRUCTURE OF RYANODINE RECEPTOR TYPE 2 DRIVES A RATIONAL DRUG DESIGN APPROACH FOR CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Abstract

Heritable mutations in RYR2 cause sudden-death predisposing arrhythmia syndromes, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), through increased sarcoplasmic reticulum (SR) diastolic Ca2+ leak. RYR2-encoded ryanodine receptor 2 is an intracellular transmembrane Ca2+ release channel in the SR of cardiomyocytes. Even on optimal medical therapy, breakthrough life-threatening arrhythmias in patients with CPVT are common. We sought to correlate identified pathogenic hotspots with key functional domains on the 3D structure of RyR2 and possible mobile binding sites to determine small molecule targets for reducing SR Ca2+ leak. We performed “signal” to “noise” (S:N) analysis in RYR2 to establish pathogenic hotspots. The minor allele frequency (MAF) of literature-derived disease-associated genetic variants at a given amino acid locus was normalized to the MAF of rare population-based variants in Genome Aggregation Database (gnomAD). Using PyMOL, a heat map overlay was generated using S:N levels and possible molecular binding pockets were identified using software PyMOL and Maestro. The highest cumulative S:N intensity of pathogenic hotspots of each domain localized on the channel domain (28.2%), helical domain 1 (25.6%), central domain (17.7%), and N-terminal domain (16.5%) of RyR2. The SPRY1 (0.498%), P1 (0.147%), handle domain (2.32%), and central U motif (8.88%) had lower S:N. Cumulative S:N intensities were nonexistent for SPRY2, SPRY3, P2, and helical domain 2. Putative small molecule binding pockets were identified in all domains while the largest number localized to the channel domain. Once binding pockets were combined with the heat map structure of RyR2, activator binding pockets of low S:N threshold in the open state of RyR2 were shown to be located heavily on the P2 domain, where CaMKII and PKA binds. The highest S:N intensity was in the channel domain with high S:N levels in other proximal domains localizing near the SR transmembrane in the lumen. Mobile binding pockets between the open and closed state of RyR2 with low S:N intensity may be alternative areas for drug targeting, especially on domains that contain binding pockets for activators of RYR2.